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Home , Aurone, Catechol oxidase, Copper protein, Polyphenol oxidase

synthase is a with PNAS PLUS hydroxylase activity and provides insights into the mechanism of plant

Christian Molitora, Stephan Gerhard Maurachera, and Annette Rompela,1

aInstitut für Biophysikalische Chemie, Fakultät für Chemie, Universität Wien, 1090 Vienna, Austria

Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved February 19, 2016 (received for review December 3, 2015) and catechol oxidases belong to the family of poly- biosynthetic processes was shown only in a few cases (1, 7–11). oxidases (PPOs). Tyrosinases catalyze the o- Recently, Sullivan (7) wondered whether there exist more PPOs and oxidation of phenolic compounds, whereas catechol oxidases that are involved in the plant’s secondary metabolism and whether were so far defined to lack the hydroxylation activity and catalyze the few so far known specialized PPOs represent only “the tip of solely the oxidation of o-diphenolic compounds. Aurone synthase the iceberg.” from Coreopsis grandiflora (AUS1) is a specialized plant PPO in- More than one decade ago, a -localized PPO, volved in the anabolic pathway of . We present, to our synthase, was identified to be a key in the anabolic aurone knowledge, the first crystal structures of a latent plant PPO, its (yellow pigments) formation in petals of Antirrhinum majus (12, 13). mature active and inactive form, caused by a sulfation of a Recently, it has been reported that aurone biosynthesis in Aster- binding . Analysis of the latent proenzyme’s interface be- aceae species (4-deoxyaurone formation) (14) differs in various tween the shielding C-terminal domain and the main core provides aspects from the 4-hydroxyaurone biosynthesis in A. majus.APPO insights into its activation mechanisms. As AUS1 did not accept homolog, termed aurone synthase (AUS1, Uniprot A0A075DN54), common substrates ( and ), the en- was assigned to be involved in 4-deoxyaurone biosynthesis in petals zyme is classified as a . However, AUS1 showed of Coreopsis grandiflora (tickseed, Asteraceae) (15, 16). As with

hydroxylase activity toward its natural (), most plant PPOs, this enzyme possesses an N-terminal BIOCHEMISTRY revealing that the hydroxylase activity is not correlated with the transit and a transfer domain and is hence pre- acceptance of common tyrosinase substrates. Therefore, we pro- dicted to be transported to the thylakoid lumen (16). AUS1 com- pose that the hydroxylase reaction is a general functionality of prises the conserved motifs of common plant PPOs—for example, a PPOs. Molecular dynamics simulations of docked substrate–enzyme thioether bridge between a and a CuA binding histidine complexes were performed, and a key residue was identified that and a (“blocker” or “gate” residue) above the active influences the plant PPO’s acceptance or rejection of tyramine. site. AUS1 exhibits an insertion in a loop region near the Based on the evidenced hydroxylase activity and the interactions and is therefore classified as a member of the group 2 PPOs (15). of specific residues with the substrates during the molecular dynam- So far uniquely, it possesses a disulfide linkage between the ics simulations, a novel catalytic reaction mechanism for plant PPOs shielding C-terminal domain and the catalytically active main core. is proposed. The presented results strongly suggest that the physi- The latent proenzyme has been shown to undergo an allosteric ological role of plant catechol oxidases were previously underesti- activation mechanism, caused by in situ-formed, highly reactive mated, as they might hydroxylate their—so far unknown—natural o- (15). Mechanistic considerations regarding the differences substrates in vivo. Significance crystal structure | tyrosinase | catechol oxidase | type III copper | mechanism Catechol oxidases and tyrosinases belong to the family of poly- phenol oxidases (PPOs). In contrast to tyrosinases, catechol oxi- yrosinases and catechol oxidases are polyphenol oxidases dases were so far defined to lack hydroxylase activity toward T(PPOs) and belong to the family of type III copper . monophenols. Aurone synthase (AUS1) is a plant catechol oxi- They are widespread among plants, fungi, and bacteria (1, 2). dase that specializes in the conversion of to aurones Plant PPOs display a high diversity, and often several PPO genes (flower pigments). We evidence for the first time, to our knowl- arepresentinoneorganism(2,3).Tyrosinases catalyze the hydrox- edge, hydroxylase activity for a catechol oxidase (AUS1) toward ylation and oxidation of monophenols (e.g., tyrosine) to o-diphenols its natural monophenolic substrate (). The presented first and the corresponding o-quinones. Due to the fact that catechol crystal structure of a plant pro-PPO provides insights into its ac- oxidases are unable to hydroxylate common tyrosinase substrates tivation mechanisms, and based on biochemical and structural (e.g., tyrosine and tyramine), they were so far defined to lack the studies of AUS1, we propose a novel catalytic reaction mecha- hydroxylation (monophenolase) activity and to catalyze solely the nism for plant PPOs. The proven hydroxylase functionality of oxidation of o-diphenols to o-quinones (diphenolase activity). AUS1 suggests that other catechol oxidases might also be in- Plant PPOs are expressed as latent proenzymes (∼64 kDa), in volved in the plant’s secondary metabolism. which the active site of the enzyme is shielded by the C-terminal domain (∼19 kDa). Presumably, the C-terminal domain is pro- Author contributions: A.R. designed research; C.M. performed research; C.M. and S.G.M. teolytically cleaved during the maturation process, resulting in a analyzed data; and C.M. wrote the paper. fully active enzyme (4, 5). The latent proenzymes can be acti- The authors declare no conflict of interest. vated in vitro by , an acidic pH, fatty acids, or deter- This article is a PNAS Direct Submission. gents (e.g., SDS) (6). PPOs are found in almost all plants and Freely available online through the PNAS open access option. have been assigned primarily to enzymatic browning reactions Data deposition: The crystallography, atomic coordinates, and structure factors have been and to the protection of organisms against biotic and abiotic deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4Z0Y, 4Z0Z,and4Z11–4Z13). stress (1, 7). However, in most cases, their individual physio- 1To whom correspondence should be addressed. Email: [email protected]. logical role and especially their natural substrates remain un- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. known. Beside aurone formation, their specific involvement in 1073/pnas.1523575113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1523575113 PNAS Early Edition | 1of10 Downloaded by guest on September 24, 2021 between tyrosinases and catechol oxidases were so far based on linker region of approximately 10 amino acids, located in the the comparison of structures of plant catechol oxidases (17, 18) vicinity of the interdomain disulfide linkage (Fig. 1C). The with structures of several bacterial (19, 20) and fungal (21, 22) C-terminal domain displays seven β-strands that form a jelly roll tyrosinases. However, the comparability of fungal and bacterial barrel motif with the four loops being arranged in a staggered with plant PPOs is very limited due to the existence of the plant manner above the active site. The latent enzyme displayed very PPOs’ bulky gate residue, which will have a general high influence low activity (toward ) and an allosteric activation (in the in substrate coordination. Recently, the first crystal structure presence of in situ-formed o-quinones) (15). Therefore, the ac- of a plant tyrosinase from walnut leaves was reported, and it was tive site of a small portion of latent enzyme molecules has to be revealed that the residues located at the entrance of the active site accessible for substrates. A loop region of the jelly roll motif as are more important for the acceptance of tyramine and tyrosine well as the loop that carries the insertion show weak or even than the previously suggested restriction of the active site (23). completely missing electron density. This demonstrates the flexi- However, the differences in the enzymatic activity and the bility of this region possibly being an entrance for the substrates mechanism of catechol oxidases and tyrosinases still remain (Fig. S2B). The interface between the catalytically active main striking questions. core and the shielding C-terminal domain is fairly large and In the following, to our knowledge, the first crystal structures composed of several hydrogen bonds, hydrophobic interactions, of a latent plant pro-PPO as well as its mature activated and an and water bridges. However, the interdomain interactions are inactive form, caused by a sulfation of a copper binding histidine, concentrated at two different locations (Fig. 2). One area is lo- are presented. Insights into the in vitro activation and the allo- cated near the linker region and the interdomain disulfide linkage, steric activation of the latent enzyme were obtained by the analysis whereas the other is located in the vicinity of the active site. The of the interdomain interactions of the proenzyme. A combination residue Ile456 is of crucial importance, as it blocks the entrance to of molecular dynamics (MD) simulations of docked natural sub- the active site and functions similar to a “plug” (Fig. 1D). This strates to active AUS1 and its hydroxylase activity toward its plug residue is located outside of the active site and on top of the natural substrate leads to the proposal of a novel catalytic reaction gate residue (Phe273). It is stabilized by its hydrophobic interac- mechanism of plant PPOs. tions with the gate residue, one CuB binding histidine (His252), and Thr253. Results Overall Structure of Active AUS1. Mature AUS1 (41.6 kDa) crystal- Copper Binding Sites of met-, oxy-, and Inactive Forms of AUS1. Active, lized in space groups P212121 and P1211, both with four monomers latent, and recombinantly expressed latent AUS1 crystallized in in the asymmetric unit, yielding crystals of comparable quality (24) the resting met-form, in which the copper ions (CuII–CuII distance, (diffracting to ∼1.6 Å; Tables S1 and S2). The enzyme’s structure ∼4 Å) are bridged by a hydroxide ion or a water molecule (Fig. possesses a very high structural similarity to the plant catechol 3A). The geometry of the met-form of AUS1 is fully consistent oxidases from Ipomoea batatas (18) [ibCO; Protein Data Bank with previously published structures (18, 23). To generate the oxy- (PDB) ID code 1BT3; rmsd of 0.81 Å between 296 atom form, crystals of recombinantly expressed AUS1 were soaked in pairs] and Vitis vinifera (17) (vvCO;PDBID2P3X;rmsdof0.79Å cryoprotectant solution containing hydrogen peroxide (24). The between 284 atom pairs) as well as to the plant tyrosinase from copper ions (CuII) of the obtained crystal structure are bridged by a J. regia (23) (jrTYR; PDB ID code 5CE9; rmsd of 0.82 Å between peroxide ion, and the Cu–Cu distance is reduced to ∼3.4 Å (Fig. 3B). 287 atom pairs). Mature AUS1 shares ∼47% sequence identity with The O–Odistanceof1.9–2.0 Å (not restrained during refinements) each of the active catechol oxidases and ∼48% with the active ty- indicates a Cu2O2 geometry that lies between the bis-μ-oxo and the rosinase. Structural divergences to these PPOs are the unique fea- μ-η2:η2-peroxo geometry, which exists in equilibrium. Interestingly, tures (15) of AUS1 (Fig. 1A and Figs. S1 and S2A): the remaining the complex exhibits a “butterfly” distortion (distortion of the Cu–O– C-terminal peptide (Asp338 to Gly452)—connected to the catalyt- O–Cu torsion angles). The Cu–O distances seem to slightly differ ically active main core by a disulfide bond (Cys206–Cys445)—and between chain A and chain B. However, the resolution (∼1.8 Å; the insertion of four amino acids (V237ANG240) that form a cavity near Tables S1 and S2) of the obtained dataset is not high enough to the active site. Both crystal forms possess homodimeric assemblies provide an unambiguous statement about the precise Cu–Odistance. of their monomers [analyzed with PISA (Proteins, Interfaces, The crystal structures of mature AUS1 (Fig. 3 C and D), pu- Structures and Assemblies)] (25), and a portion of mature AUS1 rified from the natural source, displayed an unexpected electron was found in a dimeric state by size exclusion chromatography density at the CuB binding His252. This was particularly the case (Fig. S3), evidencing that the dimer exists in solution. The biological in crystal structures from enzyme samples that exhibited a high assembly reveals a so far unknown role of the residual C-terminal portion of sulfation or phosphorylation (24), making it impossi- peptide, as it forms a main building block of the homodimeric in- ble to place a histidine-coordinated copper atom into the density. terface (Fig. 1B). The quaternary structure is stabilized by four However, the density matched very well to the density of a hydrogen bonds, 11 bridging water molecules, and several hydro- phosphohistidine or sulfohistidine. High-resolution mass spec- phobic interactions between the residues of the C-terminal peptide trometric analysis revealed that the histidine residue was sulfated of one monomer and the α-helix of the second monomer that (Fig. S4). Due to the sulfation, the histidine loses its ability to carries the C-terminal peptide. These interactions result in a buried participate in copper coordination. This leads to the loss of the surface area ranging from 1,523 to 2,323 Å2 and a free energy of CuB atom (Fig. 3D) and to the loss of enzymatic activity of the dissociation of the dimer ranging from 2.6 to 8.9 kcal/mol. The modified enzyme molecules. active sites of the dimeric monomers are facing opposite directions, due to their twofold rotational symmetry relation. Reactivity of AUS1 and Insights into the Reaction Mechanism by MD Simulations. Recently, we reported that AUS1 lacks monophenolase Overall Structure of Latent AUS1. Latent AUS1 was found to be activity (o-hydroxylation of monophenolic compounds) and displays monomeric, and the overall structure of the main core of the exclusively diphenolase activity (oxidation of diphenolic compounds latent proenzyme is almost identical to the overall structure of to the corresponding o-quinones) (15, 16). However, a modification the mature enzyme (rmsd of 0.47 Å between 338 atom pairs), of the activity assay by adding ascorbic acid as a reducing agent except for the location of one loop, which gives way for the revealed that AUS1 in fact possesses hydroxylase activity toward shielding C-terminal domain (Fig. S2B). Interestingly, this isoliquiritigenin (Fig. S5 A and B) and that consecutive reactions loop contains the characteristic insertion (V237ANG240) of the were responsible for the previously reported false-negative state- group 2 PPOs (15). The C-terminal domain begins with a short ments. In the absence of a reducing agent, the highly reactive

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Fig. 1. Overall structures of latent and mature aurone synthase. (A) Top view of mature aurone synthase. The coloring was performed according to the

secondary structure features (α-helices, green; 310-helices, yellow; β-sheets, blue), and the characteristic features of AUS1 are colored red (loop carrying the insertion V237ANG240) and magenta (residual C-terminal peptide). (B) Side view of the dimeric biological assembly of mature AUS1. The interacting residues are colored orange (monomer chain B) and magenta (monomer chain D). (C) Overall structure of latent aurone synthase. The features of the C-terminal domain are as follows: the three proteolytic cleavage sites, colored magenta and labeled as (1), (2), and (3); the linker region (orange) that connects the catalytically active domain (gray) and the shielding C-terminal domain (green); and the residual peptide of the C-terminal domain (blue) and the active site shielding Ile456 (plug residue) (red). (D) The active site is shielded by the C-terminal residue Ile456, which is responsible for the latency of the proenzyme and functions like a plug [2Fo-Fc electron density map (blue mesh) contoured at 1.0 σ].

quinoid intermediates and products polymerize and are therefore The chalcone isoliquiritigenin and the chalcone–aurone pairs of undetectable by HPLC analysis. Furthermore, the reaction product butein and lanceoletin (corresponding aurones, sulfuretin and lep- sulfuretin has been reported to be a suicide substrate for AUS1 tosidin) naturally occur in Coreopsis species [summarized in Molitor (15), for which the utilization of isoliquiritigenin strongly depended et al. (15)]. To validate obtained molecular docking results of butein to on the amount of used in the activity assay. Additionally, AUS1, two different poses were subjected to MD simulations with the the difference spectra of isoliquiritigenin differed between AUS1 met-form of AUS1. During the simulation of one docking pose, the and abTYR (Fig. S5 A and B). However, in the presence of a re- reactive B-ring of the chalcone remained in the active site, whereas ducing agent that traps the highly reactive quinoid intermediates, the A-ring was very flexible and interacted with several different resi- the difference spectra show identical absorption changes, which is dues. The other docking pose resulted in a stable substrate enzyme evidence that the differences observed in the absence of a reducing complex, where the diphenolic substrate rapidly moved deeper into the agent are related not to the hydroxylation reaction but to the cavity, which is formed by the extended loop region (V237ANG240) complex consecutive reactions. Furthermore, the enzyme showed (compare with Fig. 1A), and remained there relatively stably during the hydroxylation activity toward (Fig. S5C). In contrast to simulation time. The substrate’s atoms are almost centered tyrosinases, AUS1 did not accept tyramine and tyrosine as sub- between the two copper atoms (Fig. 4B and Fig. S6A). To mimic the strates (10 μg active AUS1; 2 mM tyramine in 1 mL total volume; substrate position during the electron transfer, the simulation was re- recordings up to 1 h; the experiments were performed with and peated with restrained Cu–O–substrate bonds (26), resulting in a without the addition of hydrogen peroxide and ascorbic acid). considerably reduced fluctuation of the unreactive A-ring due to its

Molitor et al. PNAS Early Edition | 3of10 Downloaded by guest on September 24, 2021 lanceoletin and the monophenolic substrates isoliquiritigenin and tyramine. The interactions of lanceoletin with AUS1 were almost identical with those of butein, but with additional stabilization of the methoxyl group by hydrogen bonds with Gly240 and hydro- phobic interactions arising from Val244, Ala249, and Glu248. Furthermore, the residues Gly271 and Asn271 form water bridges with the carbonyl oxygen of the chalcone. Interestingly, when the bridging water molecule is missing (water molecules in the simu- lation box were placed randomly), the highly conserved residue Glu248 is capable of directly coordinating one copper-coordinating hydroxyl group of the substrate (Fig. 4C and Fig. S6B). This residue has been proposed to participate in the water-mediated deprotonation of substrates (17, 20, 23). However, the MD simulations indicate that it might even be directly involved in substrate deprotonation (Fig. 4C). Fig. 2. Interactions between the shielding C-terminal domain and the cat- For the simulation of the monophenols isoliquiritigenin and alytically active domain of latent AUS1. The latent structure is segmented by tyramine, the oxy-form of the enzyme was used. Even without a Cu– its domains (left, catalytically active domain; right, shielding C-terminal do- O–substrate distance restraint, isoliquiritigenin remained very sta- main), and their solvent-accessible surface is presented. The interdomain ble within the cavity, with identical enzyme–substrate interactions interactions are located at two distinct areas: in the vicinity of the active site (colored red) and in the vicinity of the linker region (colored green). as in the case of the diphenols butein and lanceoletin under re- strained conditions. The distances of both copper atoms to the reactive hydroxyl group ranged mainly between 3.5 and 4.5 Å (Fig. stronger interaction with protein residues of the cavity. Butein is 4D and Fig. S6C). In contrast, tyramine rapidly left the binuclear stabilized by hydrogen bonds with the residues Asn117, Asn239, copper site of AUS1 (within 75 ps) due to attractive interactions Arg257, and Gly240 (backbone); by hydrophobic interactions with with Arg257 (Fig. 4E and Fig. S6D), which is in accordance with the the residues Asn239, Val244, Ala249, Thr253, His256, Phe273, absence of monophenolase activity of AUS1 toward tyramine. The and Ala276; and by residue Glu248 through a water bridge (Fig. characteristic insertion (V237ANG240) of AUS1 might additionally 4B and the substrate binding residues are highlighted in Fig. S1). interact with tyramine. To obtain more generalized information The unrestraint but stable docking position of butein was then about the role of Arg257, simulations of tyramine in complex with taken as a basis for the simulation of the diphenolic substrate jrTYR and with the corresponding L244R mutant of jrTYR were

Fig. 3. Copper of AUS1. The 2Fo-Fc electron density map (blue mesh) is contoured at 1.0 σ, and the anomalous Fourier difference map in A, C, and D (green mesh) is contoured at 3.0 σ.(A) Native met-copper center of recombinantly expressed AUS1. (B) Oxy-copper center of recombinantly expressed 2 2 2 2 AUS1 (μ-η :η -peroxo geometry; crystal soaked in H2O2). The oxygen atoms are slightly unsymmetrically bound to the copper atoms, and the μ-η :η peroxo complex displays a butterfly distortion. (C) Copper binding site of active AUS1 (occupancy CuB, ∼0.55). For clarity, the sulfohistidine (occupancy, ∼0.45) is not shown. (D) Inactive AUS1 (occupancy sulfohistidine, 0.9).

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Fig. 4. Reactivity and in silico-obtained substrate–enzyme complexes of AUS1. (A) Reaction scheme of the hydroxylation of isoliquiritigenin in the presence of ascorbic acid to trap highly reactive quinoid intermediates and products. (B) MD simulation snapshot of AUS1 in complex with butein. The reactive oxygen atoms at the B-ring of butein (O3, O4) are almost symmetrically located between CuA and CuB. The enzyme–substrate interactions are visualized by magenta lines (hydrogen bonding), yellow dashes (hydrophobic interactions), cyan dashed lines (π–π stacking), red dashes (cation–π interactions), and gray lines (water bridges). (C) Superimposition of snapshots obtained from different MD simulations of AUS1 with lanceoletin as the substrate. Residue Glu248 coordinates an oxygen atom of the A-ring through the formation of a water bridge (blue sticks; water bridges, gray lines) or one reactive oxygen atom (O4, B-ring) directly (white sticks; hydrogen bonding, magenta lines). (D) MD simulation snapshot of AUS1 in complex with isoliquiritigenin. (E) Three snapshots [0 ps, green (1); 44 ps, cyan (2); and 344 ps, magenta (3)] of the MD simulation of AUS1 with tyramine. Hydrophobic interactions of tyramine with Arg257 are visualized by yellow dashes.

performed. Tyramine remained within the active site of jrTYR, reveal that the main function of the interdomain linkage is the whereas it left the active site of the L244R mutant rapidly (Fig. S6E). retention of the residual peptide, as it is a main building block of These results indicate a crucial role of the residues in this position the homodimeric interface (Fig. 1B). The structure of the latent (Fig. S1) for the acceptance or refusal of tyramie as a substrate for proenzyme exhibits two domains: the catalytically active domain plant PPOs. and the C-terminal domain that is responsible for the latency of Exemplary video files of the simulation of AUS1 in complex the proenzyme. The dominating structural motif of the C-terminal with butein (Movies S1–S3) are available. domain is a jelly roll barrel. Similar motifs are also present in the shielding domains of fungal (21, 27) (C-terminal), arthropod (28) Discussion (N-terminal), and (29) (N-terminal) PPOs. This taxa-span- Overall Structures of Mature AUS1 and of the First Latent Plant PPO. ning structural homology might indicate another, so far unknown, The most relevant differences to the plant PPO homologs ibCO (17), function of the shielding domains. Due to the interdomain disul- vvCO (18), and jrTYR (23) are the characteristic four-amino-acid fide linkage of the main core with the C-terminal domain, the insertion of the group 2 PPOs and the short residual C-terminal proteolytic activation of the latent enzyme has to occur at three peptide, connected to the main core by a disulfide bond. The in- different sites (compare with Fig. S1) to result in the active en- sertion forms a binding pocket for the natural substrates (chal- zyme (15). Two proteolytic cleavage sites are exposed on the cones) of AUS1 (discussed below). The disulfide linkage was surface (1 and 2 in Fig. 1C). In contrast, the third is covered by the proposed to stabilize the C-terminal domain in the latent pro- C-terminal domain as well as by the main core but becomes enzyme (15). However, the crystal structures of mature AUS1 accessible after the proteolytic cleavage at the first two cleavage

Molitor et al. PNAS Early Edition | 5of10 Downloaded by guest on September 24, 2021 sites. This suggests, and is supported by the purification of iii) The purification has been performed independently two “semicleaved” latent forms from the natural source (15), that the times with approximately 1 y between them. The sulfohisti- proteolytic activation occurs stepwise and is targeted. The sep- dine occupancy varied from ∼0.45 (first batch, 6 kg petal aration of the interdomain interactions into two distinct areas tissue) to ∼0.9 (second batch, 9 kg petal tissue) and provides (Fig. 2) reflects specific roles of each area. The interactions close hints that the modification is either caused by sulfated com- to the linker region will be responsible for the general structural pounds of petals of C. grandiflora or occurred in vivo. integrity of the proenzyme. This is supported by the fact that Other sources of this modification might be a reaction of the interdomain disulfide linkage is also part of this area. The AUS1 with sulfated compounds within the petals of C. grandi- strength of the interactions near the active site will affect the flora during the isolation and purification procedure or an in vivo latency of the proenzyme. Furthermore, we propose that the in modification of AUS1 by a membrane-bound sulfotransferase. vitro activation (acidic activation and SDS activation) (6) as well The latter possibility is very unlikely, as a controlled in vivo PPO as the allosteric activation (15) of latent plant PPOs are pri- inactivation has not been reported so far and we do not have any marily caused by a structural rearrangement in this region. The hints that this occurred in petals of C. grandiflora. In contrast, fla- transfer of the detailed knowledge of the interdomain interac- vonoid sulfate esters are common in plants, especially in Aster- tions (Fig. S1) to other plant PPO homologs will enable the aceae species (36). Four flavonol sulfotransferases were identified targeted modulation of the kinetic properties of recombinantly in Flaveria chloraefolia and Flaveria bidentis and catalyze sequential expressed proenzymes (30). sulfation of to quercetin 3,7,3′,4′-tetrasulfate (37, 38). The active site of fungal (21, 27), bacterial (31), insect (29), Interestingly, it has been crystallographically evidenced that a direct and arthropod (28) pro-PPOs is blocked by a substrate mim- sulfuryl group transfer from p-nitrophenylsulfate to histidine, resulting icking the “placeholder” residue (tyrosine or phenylalanine, in an identical sulfohistidine as observed in AUS1, represents an originating from the shielding domain) and located within the intermediate stage in the reaction mechanism of a bacterial sulfo- active site. However, due to the presence of the bulky gate res- (39). This suggests that a direct sulfuryl transfer might also idue above the active site (phenylalanine, originating from the have taken place in the case of AUS1, as several (e.g., main core), the shielding strategy in plant PPOs has to be dif- quercetin and fisetin) (15) represent substrates for PPOs. So far, the ferent. The gate residue would have to swing away to make space preparation of samples of crude extracts (without the use of ammo- for the placeholder residue (compare with Figs. 4 and 5). As this nium sulfate) from petals of C. grandiflora, suitable for analysis by is energetically slightly unfavorable, other stabilizing interactions HPLC–ESI–MS, failed due to the extremely high amount of pig- would be necessary for compensation. The crystal structures of ments and that interfere with performed SDS/PAGE the first latent plant pro-PPO reveal that plant PPOs overcome analysis. However, the development of effective tyrosinase inhibitors this issue by a smaller hydrophobic plug residue (Ile456 in AUS1; has become increasingly important in the cosmetic, medicinal, and valine, , and isoleucine in other plant PPOs) located agricultural industries for application as antibrowning and depig- above the gate residue and outside of the active site (Fig. 1D). menting agents, and the inactivation of a PPO by a sulfonated sub- strate would have a high impact in this field of research. Copper Binding Site of AUS1. The reactive oxy-form of AUS1 ex- hibits a butterfly distortion (Fig. 3B) similar to the distortion found Reactivity, Enzyme–Substrate Simulations, and Reaction Mechanism in the oxy-form of Streptomyces castaneoglobisporus tyrosinase of Plant PPOs. AUS1 is a catechol oxidase type of PPO, as no sc ( TYR) (31). Because no indications of a distortion were found reactivity toward the common tyrosinase substrates tyramine and oxy in ligand field molecular dynamical simulations of the -form of tyrosine was detectable. However, AUS1 showed monophenolase tyrosinase (32), it has been suggested that the distortion in scTYR activity toward its natural substrate isoliquiritigenin (chal- might be caused by a hydrogen bonding interaction of the place- cone) and the flavonol kaempferol (Fig. S5). A similar reactivity holder residue (tyrosine) with the oxygen atoms. However, the has been reported for catechol oxidase from Aspergillus oryzae oxy-form of AUS1 indicates that the butterfly distortion is a gen- (aoCO4) (40). This fungal PPO showed activity toward amino- eral characteristic of the oxy-form of PPOs, as AUS1 does not phenol and guaiacol but not toward tyrosine. Historically, the possess a placeholder residue that might cause this distortion. definition that catechol oxidases lack the hydroxylase activity was Irreversible inactivation of tyrosinase from Agaricus bisporus primarily based on the acceptance of common tyrosinase sub- (PPO3; abTYR) has been reported to occur by incubation with strates (e.g., p-cresol, tyrosine, tyramine) and not on the hy- sulfite ions (33). The authors evidenced the sulfation of a CuB- droxylation of their natural substrates (or at least a wider range coordinating histidine and speculated that the sulfation (ac- of potential substrates). Due to the evidenced hydroxylase ac- companied by the loss of the CuB atom) presumably occurred at tivity of AUS1 and aoCO4 (40), the question arises as to whether His263 (His256 in AUS1). The crystal structure of AUS1, how- e this definition is still valid. There is no doubt that catechol oxi- ever, reveals that the sulfuryl group is transferred to the N atom dases are incapable of hydroxylating tyramine or tyrosine, how- of His252 (His259 in PPO3; abTYR). The consistent discovery of ever AUS1 and aoCO4 reveal that they might hydroxylate this inactivation in both PPOs might indicate an infiltration of various other monophenols (e.g., their potential natural sub- sulfite ions during the purification procedure of AUS1. However, strates). Furthermore, the comparison of the crystal structures of there exist several matters that evoke doubts about this: plant catechol oxidases (PDB ID codes 2P3X and 1BT3) with the i) The chemicals used to purify AUS1 are not only commonly structure of a plant tyrosinase (PDB ID code 5CE9) did not used in protein purification, the identical chemicals were also reveal relevant structural differences except in the potential used in the purification of tyrosinases from J. regia (34) and substrate binding residues (23). In this study, we identified a A. bisporus (35) and catechol oxidase from V. vinifera (24) in substrate binding residue (by performing MD simulations, dis- either similar or even identical methods. Mass spectrometric cussed below) that is able to stabilize or destabilize the tyrosinase analyses [HPLC–ESI–MS (HPLC electrospray ionization mass substrate tyramine within the active site of plant PPOs. This spectrometry) of digested protein samples and ESI–Q-TOF– supports the presumption that no general structural limitation MS (electrospray ionization quadrupole time-of-flight mass regarding the hydroxylase activity of plant PPOs exists, except the spectrometry) of intact protein samples] were performed prerequisite of a substrate stabilization in a way that a hydrox- routinely, and none of the PPO samples exhibited any in- ylation reaction can occur. However, further investigations will dication of such a modification (34, 35). be necessary to prove a putative general hydroxylase activity of ii) The latent form of AUS1 also did not display any modification. PPOs. Therefore, we suggest using a substantially broader range

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1523575113 Molitor et al. Downloaded by guest on September 24, 2021 PNAS PLUS BIOCHEMISTRY

Fig. 5. Proposed catalytic reaction mechanism of plant PPOs. (A) Monophenolase cycle: The monophenolic substrate is guided to the binuclear copper site 2 2 [oxy-form, Cu(II)2O2, μ-η :η peroxide complex] by displaced π–π interactions with the tilt out gate residue Phe273 and cation–π interaction with the CuB binding histidine His256 (M1). When the substrate binds to the copper atoms, hydrophobic interactions with the substrate’s para-substituent become im- portant additionally. The reactive substrate oxygen atom binds equally to both copper ions (M2), and the peroxide ligand is transferred to an inverse butterfly distortion (ligand field molecular dynamical simulations, the Cu–O–O–Cu torsion angles of the peroxo ligand fluctuate by ±20°) (32). Nucleophilic attack of

the Cu2O2 moiety by the substrate results either in an o-diphenolic product and the met-form [M4D, Cu(II)2OH] or in an o- and the deoxy-form [M4Q, Cu(I)2]. During product release, the gate residue swings back to its preferred position. Finally, oxygen uptake closes the catalytic cycle (M5). (B) Diphenolase cycle: The principles of the diphenolase cycle are similar to the monophenolase cycle described in A. The reactive substrate oxygen atoms bind equally to both copper ions of the met-form (D1, D2), and the o-quinone is released after electron transfer to the binuclear copper site resulting in the deoxy-form (D3). Oxygen uptake results in the oxy-form (D4) and substrate binding results in an inverse butterfly distortion in the transition state (D5). The catalytic cycle is closed by the release of an o-quinone resulting in the met-form of the binuclear copper site.

of compounds (including the class of flavonoids—e.g., chalcones, toward compounds that were previously reported to be tyrosinase , flavanols) in activity assays for plant PPOs. Furthermore, inhibitors but in fact are hydroxylized and oxidized by tyrosinase we suggest varying the conditions by adding reducing agents (to (41) [e.g., arbutin and isoeugenol (42), phloridzin and (43)]. avoid or enzyme inactivation) or hydrogen perox- The very specific interactions observed during the MD simu- ide. The latter is useful not only in omitting the lag phase of the lations of AUS1 with the naturally occurring chalcones strongly hydroxylation reaction but also in detecting hydroxylase activity support the hypothesis that the crystallized enzyme represents a

Molitor et al. PNAS Early Edition | 7of10 Downloaded by guest on September 24, 2021 specialized enzyme involved in aurone biosynthesis (15, 16). strates that in the long term the classification and the nomenclature Furthermore, the proposed importance of the characteristic in- of plant PPOs will have to change to avoid misunderstandings. sertion of the group 2 PPOs in substrate binding (15) is confirmed As a consequence of the evidenced hydroxylation activity of (Fig. 4B and Movies S1–S3). However, although the substrates are AUS1 toward its natural substrate in combination with the con- strongly stabilized, it should be mentioned that the diphenols do sistent proposed novel reaction mechanism, another complexion is not “have” to bind within this binding pocket. PPOs accept an put on the function of plant PPOs. So far, the role of plant PPOs enormous variety of diphenolic substrates (e.g., chlorogenic acid, has primarily been assigned to enzymatic browning reactions, with 4-tert-butylcatechol, 3,5-di-tert-butylcatechol), suggesting that even only few exceptions [e.g., (12, 13) and aurone unspecific binding to the copper center can result in product synthase (15, 16)]. However, we demonstrated that the mono- formation. Apart from the characteristic insertion of AUS1, the phenolase activity of plant PPOs seems to depend only on whether involvement of several residues at the copper binding sites (Fig. 4B the monophenol can be stabilized within the active site or not. and Fig. S1) in substrate coordination will be transferrable to There is no doubt that the hydroxylase activity represents the other plant PPOs. primary functionality of tyrosinases (e.g., in the hydroxylation of It has been shown that in a bacterial tyrosinase (from Bacillus tyrosine in the biosynthetic pathway). If catechol oxi- megaterium; bmTYR) the substrates bind to the CuA atom, and dases also possess hydroxylase activity, it seems reasonable that therefore, it was concluded that the monophenols will perform a the above statement is also true for this type of enzyme. As a substrate rotation during the hydroxylation reaction (20). The consequence, it could be assumed that the catechol oxidases are authors proposed that the substrates will bind to the same copper involved in anabolic pathways by hydroxylating their—so far atom in plant catechol oxidases. Based on these assumptions, it unknown—natural substrates. Hence, it can be concluded that was further concluded that the hydroxylation reaction is im- the identification of the few plant PPOs involved in the sec- possible in plant catechol oxidases, as the bulky gate residue in ondary metabolism does indeed represent just the “tip of the ” combination with the rigidity of the CuA site (thioether bond) will iceberg (7). hinder the substrate rotation. However, the proposed mechanism There is no doubt that there will be a long interdisciplinary is incompatible with the hydroxylation activity of jrTYR (34, way to go (including metabolomics and transcriptomic and pro- 44) as well as the observed hydroxylation activity of AUS1, as teomic analyses) until the role of plant PPOs is clarified, partic- both possess a bulky gate residue and a thioether bond. Fur- ularly with regard to their complex diversity. The substrate thermore, no explanation for the lack of monophenolase activity binding residues (compare Fig. 4 and Fig. S1) around the CuA of aoCO4 toward tyrosine as a substrate (40) was given. and CuB binding sites differ considerably within the family of Naturally, classical MD simulations are not suitable to obtain PPOs (2, 3), indicating the occurrence of individual natural sub- information about the geometry of the substrate–copper binding strates. Therefore, the upcoming challenges will be to cluster the scenario, as they do not take into account quantum mechanical PPOs according to their substrate binding residues, to identify the – potential natural substrates (or substrate scaffolds), and to vali- effects or the Jahn Teller distortion. However, the strong hy- ’ drophobic interactions of the substrate with the gate residue date the involvement of the corresponding PPOs in the plants (Phe273) and His256 direct the B-ring to the active site, where the secondary metabolism. ’ substrate s o-diphenolic oxygen atoms are almost equally positioned Methods between both copper ions (Fig. 4 B and C and Fig. S6). The posi- Protein Purification, Crystallization, Data Collection, Structure Determination, tions of the oxygen atoms are in a way comparable with the position and Refinement. The protein purification of active and latent AUS1 from of the butterfly distorted oxygen atoms of the oxy-form, demon- petals of C. grandiflora is described in Molitor et al. (15), and the purification strating the general possibility of this coordination. Further- of recombinantly expressed AUS1 is described in Kaintz et al. (16). The more, the monophenol isoliquiritigenin is stabilized in a position crystallization of the obtained enzyme samples and the data collection, us- that allows the hydroxylation in the ortho position to the copper- ing synchrotron radiation, of the obtained crystals have been described in coordinating hydroxyl group (Fig. 4D). These observations in Molitor et al. (24). The corresponding data collections of the presented combination with the detected hydroxylation activity of AUS1 crystal structures were performed using synchrotron radiation at 100 K (PDB ID — λ = — (Fig. S5)andjrTYR (23, 34, 45) led us to propose a novel code 4Z0Y P14, PETRA III, EMBL, DESY; 1.23953 Å; PDB ID code 4Z0Z I04-1, DIAMOND; λ = 0.9173 Å; PDB 4Z11—ID23-1, ESRF; λ = 0.972499 Å; PDB 4Z12— mechanism for the hydroxylation and oxidation of phenolic sub- P11, PETRA III, DESY; λ = 1.0247 Å; PDB 4Z13—I04-1, DIAMOND; λ = 0.9173 Å). strates by plant PPOs (Fig. 5). In this mechanism, no substrate Automated refinements of all models (obtained as described below) were rotation is required, as the substrate is coordinated equally to both performed by phenix.refine from the PHENIX suite (47) followed by manual copper ions. This removes the contradiction of the previously structure completion and correction using COOT (48).

proposed mechanisms with the experimentally proven mono- Initial phases for the active AUS1 form (space group P212121)wereobtained phenolase activities of plant PPOs. Furthermore, the MD simu- by molecular replacement using the BALBES webserver (49), which used the lations of tyramine in the active site of AUS1, jrTYR, and the crystal structure of catechol oxidase from I. batatas (PDB ID code 1BT3, se- L244R mutant of jrTYR strongly suggest that the residue at the quence identity ∼47%) as a search model. The refined structure of AUS1 was position of Arg257 (compare with Fig. S1) is one of the key res- then used as a search model for structure determination of the remaining obtained datasets by molecular replacement using PHASER (MR) from the idues for the acceptance of tyramine as a substrate for plant PPOs. PHENIX program suite. The structure of the latent AUS1 form was obtained by It should be noted that the interactions will be different in fungal automated model building using AUTOBUILD from the PHENIX suite, after and bacterial PPOs, as they do not possess a bulky gate residue placing the refined catalytically active main core with PHASER (MR) in the and the substrates are bound to the CuA atom (20). However, asymmetric unit. Restraints for the polyoxometalate hexatungstotellurate (VI), during MD simulations the classical tyrosinase substrate tyramine used as a cocrystallization agent for recombinantly expressed AUS1 (24, 50), is destabilized in the active center of plant PPOs by an arginine in were obtained with phenix.reel and phenix.elbow as previously reported (27, this position (AUS1, L244R mutant of jrTYR), whereas it is sta- 51, 52) using the crystal structure of hexatungstotellurate (VI) (53). The quality bilized by a leucine in this position (jrTYR). It has been reported of the models was validated by MOLPROBITY (54). All models were refined to excellent stereochemistry without any Ramachandran outliers, and the re- recently that vvCO, possessing a lysine in this position, showed spective refinement statistics are presented in Tables S1 and S2. weak activity toward tyrosine and tyramine, and vvCO was The models were deposited in the PDB (www.rcsb.org) as ID codes 4Z0Y, therefore classified as a tyrosinase (46). However, a comparison of 4Z0Z, 4Z11, 4Z12, and 4Z13. the kinetic data of vvCO and jrTYR indicates that tyramine will presumably not be the natural substrate of vvCO [Km(vvCO) = Protein Digestion and HPLC–ESI–MS Measurements of Inactive AUS1. The ex- 7.7 mM (46); Km(jrTYR) = 0.274 mM (23)]. This example demon- periments were performed by the company Proteome Factory. The protein

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1523575113 Molitor et al. Downloaded by guest on September 24, 2021 sample was denatured by incubation in 8 M urea for 30 min at room temperature. box (10 Å edge distance) of TIP3PBOX water molecules. The charge of the PNAS PLUS For reduction, a final concentration of 5 mM Tris(2-carboxyethyl)phosphine system was neutralized by replacing water molecules by the respective num- was added, followed by incubation for 20 min at room temperature. Then ber of sodium ions. Energy minimization was performed with the steepest step iodoacetamide was added to a final concentration of 10 mM. After in- algorithm with a maximum of 3,000 steps and an energy tolerance of 1,000 cubation for another 20 min in the dark, the samples were diluted to 0.8 M kJ·mol−1·nm−1. After convergence, the system was equilibrated in two phases: urea and subsequently digested by two endoproteases (trypsin and AspN) NVT ensemble (isothermal-isochoric: constant number of particles, volume, and with an enzyme:protein ratio of 1:50 (wt/wt) according to the Proteome temperature) and NPT ensemble (isothermal-isobaric: constant number of Factory’s protein digestion standard operating procedures. The acidified particles, pressure, and temperature), each for 100 ps at 300 K. MD simulations peptide populations were applied to nanoLC–ESI–MS (LTQ-FT, Thermo Fin- were carried out by applying periodic boundary conditions. The LINCS (Linear nigan) analyses using a 60-min nanoLC gradient (Agilent 1100 nanoLC system) Constraint Solver) algorithm was used to constrain all bond lengths, and the with solvent A [0.1% (vol/vol) formic acid in 5% (vol/vol) acetonitrile] and particle-mesh Ewald method was used to calculate long-range electrostatic solvent B [0.1% (vol/vol) formic acid in 99.9% acetonitrile]. interactions. The Berendsen coupling algorithm was used for temperature, and ± The mass accuracy was better than 3 ppm for MS data and 0.02 Da for the Parrinello–Rahman coupling algorithm was used for pressure control. 2 2 MS data. MS data analyses were done using MASCOT (Matrix Science), After the two equilibration runs, a 5-ns simulation was carried out with, whereas the MS data were analyzed using Qualbrowser (Thermo Finnigan). unless otherwise stated, no position restraints. The induced modification carbamidomethylation (C) was allowed during MS2 data searches, along with possible modifications such as oxidation (M), Structural Analysis and Graphical Presentation. The quaternary structure of deamidation (NQ), and phosphorylation/sulfation (H). The precursor show- the obtained crystal structures was analyzed with PISA (Proteins, Interfaces, ing the predicted modification and a strong neutral loss signal was subject to Structures and Assemblies) (25). Protein interfaces and ligand interactions dedicated MS3 experiments. were analyzed and visualized with LigPlot+ (61) and PLIP (Protein–Ligand Interaction Profiler) (62). Molecular graphic images and movies were gen- Molecular Docking and MD Simulations. The structures of active AUS1 (PDB ID erated with PyMOL (www.pymol.org/) and VMD (Visual Molecular Dynamics) code 4Z0Y) and jrTYR (PDB ID code 5CE9) were prepared for molecular (63), respectively. The secondary structure features were assigned using DSSP docking studies and MD simulations by the addition of missing side chain (Dictionary of Secondary Structures of Proteins) (64). atoms using COOT and the removal of the residual C-terminal peptide of AUS1. Molecular docking of butein was performed with AutoDock VINA (55) by the use of the deoxy-form of active AUS1 and setting the gate residue ACKNOWLEDGMENTS. We thank Kristina Djinovic-Carugo and Georg Mlynek [Max F. Perutz Laboratories (MFPL), Vienna Biocenter] for their kind support PHE273 as a flexible residue. The search exhaustiveness was set to the maximal – during the early stages of this research. We thank beamline scientists Elspeth value of 2,000 (default 8). MD simulations of obtained enzyme substrate Gordon (ESRF ID23-1, mx1450), Anja Burkhardt (DESY P11, I-20120633 EC), and complexes were performed with GROMACS (56) version 4.6.7 package apply- Alice Douangamath (Diamond Light Source I04-1, MX8476) for their generous

ing the AMBER99SB force field (57). The ligand topologies were generated support during the allocated beam times. We give special thanks to Gleb Bour- BIOCHEMISTRY using the SWISSPARAM webserver (58). RESP (restrained electrostatic poten- enkov and Victor S. Lamzin (DESY/EMBL) for the opportunity of data collection tial) charges were obtained from the RED webserver (59) by applying the HF/6– at beamline P14 during “European School for Macromolecular Crystallography ” 31G* level of theory (Gaussian09) to calculate the electrostatic potential. For (ESMAX) 2012. For cultivating C. grandiflora and for taking care of the plant the simulation of o-diphenols in complex with AUS1, the met-form was used fields, we thank the Horticultural Department of Molecular Systems Biology, UZA1-Glashaus1, University of Vienna—in particular Thomas Joch and Andreas and new residues were defined for the copper atoms, the copper-coordinating Schröfl—and the gardeners of the experimental garden Augarten—especially , and the thioether forming cysteine, according to Bochot et al. (60). Miroslav Crep and Erich Wagner. We also thank Aleksandar Bijelic, Matthias For the simulation of monophenols in complex with AUS1, the oxy-form was Pretzler, and Ioannis Kampatsikas for valuable discussions concerning this used and parameters of the dinuclear copper site were adopted from Deeth work. The research was funded by the Austrian Science Fund (FWF): P25217 and Diedrich (32). The protein–ligand complex was solvated in a dodecahedral and the Deutsche Forschungsgemeinschaft (Ro 1084/8-1).

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